Progressive dilution series apparatus



Oct. 22, 1968 T m 3,406,635

PROGRESSIVE DILUTION SERIES APPARATUS Filed Jan. 17, 1966 2 Sheets-Sheetl F i g Leif $612929 1 N VEN TOR.

Oct. 22, 1968 Filed Jan. 17, 1966 L. TYBRING 3,406,635

PROGRESSIVE DILUTION SERIES APPARATUS 2 Sheets-Sheet 2 w ,J /z' 4 Z6 50.2) j S United States Patent PROGRESSIVE DILUTION SERIES APPARATUS LeifTybring, Gentofte, Denmark, assignor to Lovens Kemiske FabrikProduktionsaktieselskab, Ballerup,

Denmark, a firm Filed Jan. 17, 1966, Ser. No. 521,204 Claims priority,application lgenmark, Jan. 18, 1965, 2 5/6 7 Claims. (Cl. 103-149)ABSTRACT OF THE DISCLOSURE The apparatus consists of a rotating orperistaltic working piston pump inserted into a conduit for pumpingvolumetric quantities of a solution in one or the other directionthrough a conduit, one end of which forms a connection to a reservoirfor diluent while the other end is free to be lowered into a containerfor basic or diluted solution, the pump being provided with means forterminating the pumping after a measured quantity has been transportedtowards the end of the conduit to be lowered into the basic or dilutedsolution and is a predetermined number of times greater than whentransporting the liquid towards the reservoir.

In laboratories it is often required to produce a progressive-dilutionseries, i.e. a number or series of solutions in which a startingmaterial is present in concentrations that are consecutive members of ageometrical progression. From a substance having the startingconcentration A it may thus be desirable to produce aprogressive-dilution series, the members of which have theconcentrations A, A- 10- A- l0" A- 10" and A-l0 respectively, n being aninteger. It is not unusual that progressive-dilution series are requiredof the order of 5 to 10 members and in some kinds of experimental workthe production of such series is a very frequently occurring task.

The usual way of producing progressive-dilution series is by using apipette, which is rather laborious. It consists in extracting with asmaller pipette a predetermined portion of the basic solution, afterwhich a portion of diluent is extracted from a suitable reservoir bymeans of a pipette, the volume of which is p1 times the volume of thefirst one, if the rate of dilution is to be p. The measured portion ofthe basic solution is then mixed with the measured portion of thediluent. Then a measured portion of the solution thus produced isextracted by means of another pipette of the small size, and anothermeasured amount of the diluent is extracted with the larger pipette,after which the liquids are mixed. Again, a portion of the thus producedmixture is extracted by means of still another pipette of the smallervolume, and a portion of the diluent is extracted by means of the largerpipette, the liquids are mixed and so on. Thus, for each step in thedilution series a further clean pipette of the smaller volume must beused and in some cases also a further pipette for each portion of thediluent to be extrac'ed. That is the case if complete emptying of thelarger pipette is achieved by using it for the stirring. Alternatively,the larger pipette used for measuring the diluent may be kept clean, butin this case special care must be taken with regard to emptying it sothat the same dilution ratio is obtained each time, which takes aconsiderable time. Anyhow, for each progressive-dilution series a greatnumber of pipettes is necessary, which pipettes must be cleansed anddried and perhaps sterilized before they can be used anew.

The invention aims at providing an apparatus for the production ofprogressive-dilution series which apparatus 3,406,635 Patented Oct. 22,1968 is simple and by which the above-mentioned drawbacks are avoided.

The apparatus according to the invention consists in a rotatingly orperistaltically working piston pump inserted into a conduit and designedfor pumping volumetrically quantities of a solution in one or the otherdirection through the conduit. One end of the conduit forms a connectionto a reservoir for diluent while the other end is free to be loweredinto a container for'basic or diluted solution. The pump is providedwith means. for terminating the pumping after a measured quantity hasbeen transported, which quantity when the pump works to transport theliquid in a direction towards the end of the conduit adapted to belowered into the basic or diluted solution, which end is above and inthe following designed as the free end, is a predetermined number oftimes greater than when the pump is working to transport the liquid in adirection towards the reservoir for the diluent. The volume transportedin the latter case between the termination points thus defined will bedesigned in the following as the unit volume.

The apparatus works in the following way: When the conduit is filledwith the diluent and one end of the conduit is in connection with areservoir for the diluent, and the other end, the free end, is loweredinto a container with the basic solution, one or more unit volumes arefirst sucked into the conduit, whereby a corresponding amount of diluentis simultaneously delivered to the reservoir containing the reserveportion of diluent. Thereupon the pumping terminates and the free endthrough which the basic solution has been sucked in, is transferred to acontainer for receiving diluted solution. Now pumping is re versed andthe predetermined number of unit volumes is pumped into said container.Firstly, the basic solution just sucked up leaves the conduit and then anumber of unit volumes of the diluent corresponding to the desired totalvolume. On being pumped out the two liquids mix and before the free endof the conduit is removed from the just formed diluted liquid and byreversing and starting the pump one or more unit volumes of the mixtureare sucked into the conduit, whereupon the end of the conduit istransferred to an empty container serving to take up the next dilutionstep of the series, the pump is reversed and the predetermined number ofunit volumes is given off. The process continues in this manner until aseries of liquids with increasing degree of dilution has been obtained,the last of which has the highest degree of dilution required for theexperiment in view.

As it will be seen it is possible by using the apparatus to achieve thatthe mixing with diluent owing to the pump pressure takes placespontaneously, that the apparatusdoes not need any separate rinsingduring the process of producing a series of progressive dilution, as thefree end through which the basic solution or the last formed dilutedsolution is taken up will always be rinsed properly by the passage ofthe diluent through it, that the pipettation takes place automaticallyand that the apparatus is always ready for use as long as the samediluent is used. If a different diluent is to be used for anotherprogressivedilution series the apparatus is readily rinsed with the newdiluent by just pumping the same through the conduit.

Furthermore the apparatus is very Well suited for automatic productionof progressive-dilution series as the containers for the diluted liquidmay be placed on a conveyor or a turning table, so that anothercontainer automatically replaces the former one after each dilutionoperation.

In a preferred embodiment of the apparatus according to the inventionthe volume of the conduit, including the inserted pump volume, is largerthan the unit volume. Hereby it is obtained that the basic liquid ordiluted liquid sucked up fills only part of the conduit and that nothingof these liquids escape at the other end which would result in that thesaid other end of the conduit had to be transferred to a container forbasic or diluted liquid thus pumped out, which would be impractical.Likewise, if the volume of the conduit, including the inserted pumpvolume was to have the same size as the unit volume, a diffusion of thediluted material into the diluent might occur, whereby said diluentwould be contaminated and part of the substance contained in themeasured amount of liquid might be lost, whereby theprogressive-dilution series would be unreliable. It is even preferablethat the volume of the conduit is several times that of the unit volumewhich permits more than one volume to be taken up and consequently avariety of dilution rates can be obtained. In many cases it is alsopreferable that only the foremost part of the conduit, i.e. the partlying between the diluent and the pump space, is used for taking up aunit volume of the basic liquid or the diluted liquid, or for taking upa few unit volumes. Hereby a reliable cleaning of the apparatus duringthe working is ensured.

The apparatus according to the invention is very simple and cheap whenthe pump is a rotating piston pump of the valveless type, adapted forbeing driven with alternate directions of rotation, preferably by meansof an engine, the direction of rotation of which can be shifted. Asexamples of rotating piston pumps of the valveless type which can beused according to the invention, may be mentioned a rotary vane pump, agear pump or a flexible-hose pump. On the other hand, nothing preventsthe use of a piston pump with valves, but these must be reversible sothat the pump can work both ways. By using a valveless pump, however,certain drawbacks are avoided such as faults owing to possible insecureclosing of the valves, and that the valves owing to their complicatedform may retain impurities or undiluted liquid and thus causecontamination. or inaccuracy in the progressive-dilution series. In thecase of microbiological works the valves may furthermore give rise toinfection from one series to the other. When the piston pump isvalveless the measured volume is determined by the rotating angle of therotor and the pumping direction is controlled by the direction ofrotation of the rotor. Both are easily adjusted either mechanically orelectrically.

As compared with other rotating piston pumps of the valveless type aflexible-hose pump has the advantage that the piston moves on theoutside of the hose and the closing operation is performed merely bycompressing said hose. Thus, it is only the hose which comes intocontact with the measured and transported liquid and the pump space andthe conduit may form a continuous jointless system with smooth wallswhich is exceedingly easy to keep clean by rinsing. As mentioned above,when the apparatus is working a constant rinsing with the diluent takesplace in connection with each dilution operation, and consequently apump of the last-mentioned type needs practically no special cleaning.

The apparatus according to the invention where the pump is a rotatingpiston pump of the valveless type, for instance a flexible-hose pump,may preferably be so constructed that the rotor shaft carries or is inconstant transmission engagement with a cam wheel having one or morecams serving to discontinue the rotation of the rotor shaft aftertransportation of a unit volume. Hereby a reliable measuring of the unitvolumes is obtained. As is well known a flexible-hose pump has deadpoints; by making the moment for cutting off the rotation of the rotorshaft coincide with the moment when the piston passes one of the saiddead points, it is possible to obtain an extraordinary exactitude ofmeasuring.

In order to make such an apparatus particularly easy to manage accordingto the invention the rotor shaft may be in transmission engagement witha second cam wheel by means of a one-way connection, for instance aratchet connection; said second cam may be provided with one or morecams which serve to discontinue the rotation of the rotor shaft aftertransportation in the direction towards the container for diluted liquidhas taken place, the transmission ratio between the first and the secondcam wheel being decided by the dilution ratio and the ratio between thenumber of cams on the two cam wheels. Hereby the apparatus can be workedsolely by starting the rotor shaft in one or the other direction, saiddirection deciding whether the rotor shaft moves in steps correspondingto the measuring of a unit volume or in steps corresponding to themeasuring of a total volume which is a predetermined number of timeslarger than the unit volume.

In the following the invention is described in greater detail bydescribing an apparatus which is an embodiment of the invention,reference being made to the drawing, in which FIG. 1 shows theembodiment in question, seen from above, together with a reservoir fordiluent and a container for basic liquid or diluted liquid,

FIG. 2 the same apparatus, partly in longitudinal section,

FIG. 3 horizontal section through the apparatus in FIG. 2, along theline IIIIII, and

FIG. 4 a diagram showing the electric connections in the apparatusaccording to FIGS. 1-3.

In FIGS. 1, 2 and 3 the reference number 1 is a house with cylindricalrear wall 2, a plane front and a flat roof 3. In continuation of thecylindrical rear wall there is an edge reaching up above the roof, saidedge forming support for a conduit consisting of a tube 6 of elasticpliable material, such as for instance rubber or plastic. One end of thetube 6 is connected to the liquid in a reservoir 29 for the diluent, theother, which is provided with a nozzle 31 is lowered into a container 30which may either be a container containing the basic liquid from which aprogressive-dilution series is to be produced by means of the diluent inthe reservoir 29, or an empty container, in which diluted liquid is tobe filled in and from which a measured amount of the thus diluted liquidis again to be sucked up for use in the next step in theprogressivedilution series.

4 is a rotor of the kind known from flexible-hose pumps which on theends of motor arms carries rollers 5, which during the passage along thecylindrical rear wall 2 compresses the tube 6 so that it closes securelyagainst passage of liquid. The rotor arms 4 with the rollers 5 thus formthe pistons of the flexible-hose pump. The rotor is carried by a shaft9, which is driven by a motor 7 through a gearbox 8. The shaft issupported in the roof 3 by a bearing 13. The rotor shaft 9 carries aspur-gear wheel 11 and a cam wheel 10 which on its circumference has acam 24. The gear wheel 11 and the cam wheel 10 are mounted on the rotorshaft 9 in fixed connection therewith. During the rotation of the rotorshaft the cam 24 gets into engagement with the contact of a micro-switch26 of the normally-closed type, which opens when its contact engages thecam 24. The gear wheel 11 is in constant engagement with another gearwheel 17, which is freely rotatable on a shaft 14 parallel with therotor shaft and mounted in journals 15 and 16 carried on bracketsmounted on the walls but not shown in the drawing. The spur-gear wheel17 carries adjacent to the circumference thereof a pin 19 upon which aratchet 2 0is rotatable. The ratchet 20 is in engagement with a ratchetwheel 18 mounted on the shaft 14 in fixed connection therewith. Byrotating the gear wheel 17 in one direction the shaft 14 mustaccordingly follow driven by the ratchet 20, whereas when the gear wheel17 is turned in the opposite direction, the shaft 14 remains unmoved,kept still by friction means not shown on the drawing. The shaft 14further carries a worm drive 21 in fixed connection therewith and inengagement with a worm gear 22 carried on a shaft 41. The shaft 41further carries a cam wheel 23 with a cam 25. The worm gear 22 and thecam wheel 23 are in fixed connection with the shaft 41. A micro-switch27 of the normally-closed type is mounted on a bracket not shown in suchposition that it gets in engagement with the cam 25, when the cam wheel23 is rotated. By such engagement the micro-switch 27 opens. In someconvenient place, for instance as shown, a relay 28 having a coil, anarmature and a closing and a shifting contact as later on described, areplaced in the housing 1.

In the following the ratchet and the ratchet wheel are assumed to beoriented in such a manner that the shaft 14 is rotated when the rotorshaft 9 is moved in clockwise direction in FIGS. 1 and 3, i.e. whenliquid is pumped from the reservoir 29 to the container 30.

The electric connections are not shown in FIGS. 1-3 but illustrated inthe diagram shown in FIG. 4.

In this diagram the reference number 32 shows a phase line of anelectric current distribution network. In the phase line is inserted aglow discharged lamp 33 and a main circuit breaker 44. From this anelectric wire 45 is carried to the switch 27, a connection 46 to theother switch 26, a connection 47 to a foot-operated switch 39 of thenormally-open type, which can be closed by pressing the pedal and afurther connection 48 to another footoperated switch 40 of the samekind. The relay 28 mentioned above consists of a coil 38, which isactivated through the connection 53 and an armature having two contacts34 and 57, the former closing a contact 35 when activated, the latterbreaking a contact 36 and closing a contact 37 when activated. From thefoot-operated switch 40 a connection 49 is carried to the movablecontact 57 from which a connection 50 is carried to the switch 26. Fromthe foot-operated switch 39 a connection 51 is carried to the fixedcontact 35 and from the movable armature contact 34 a connection 52 iscarried to the switch 26. From the switch 27 a connection 58 is carriedto the motor 7 and from the fixed contact 37 a connection 55 is carriedto another terminal of the motor 7. A third terminal of the motor 7 isconnected with the other end 54 of the phase line 32 and the sameapplies to the spool 38 which is connected to the motor 7 through aconnection 54.

When the foot-operated switches 39 and 40 are opened, the motor 7 hascurrent though the connections 45 and 58 if the switch 27 is closed, andin this case the motor continues running clockwise until this switch isopened by the cam 25 of the cam wheel 23 getting in engagement with themicroswitch 27. When hereby the motor has been stopped and thefoot-operated switch 39 is pressed, the coil 38 is activated through theconnections 51 and 53 and thus pulls its armature with the movablecontacts 34 and 57. Hereby the said contacts 34 and 57 are displaced tothe right and close the current though the connection 46, thenormallyclosed switch 26, the fixed contact 37 and the connection 55,which causes the motor 7 to start and rotate the rotor shaft inanticlockwise direction. If the foot switch 39 is then immediatelyclosed the motor continues running until the cam 24 breaks themicro-switch 26. The relay 28 is thereby inactivated. After this themotor is able to restart solely by pressing either the foot switch 39 orthe foot switch 40. If the foot switch 39 is pressed, the same willhappen as above described, viz. the motor gets current through theconnection 55 and accordingly rotates in such a direction that the rotormoves in anti-clockwise direction. Thereby a unit volume is sucked infrom the container 30 and the motor stops again after having moved therotor one turn. This can be repeated a few times, more or less dependenton the volume of the conduit 6, since the amount of liquid sucked inmust not 'be allowed to fill the said conduit or get too close to theend of the conduit connected with the diluting agent contained in thereservoir 29.

If, on the other hand, the foot switch 40 is pressed, although the motorwill start, the relay 28 is not activated. Accordingly, the currentpasses the connections 48 and 6 49, the contacts 57 and 36, theconnections 56 and 58 to the motor 7. The motor now runs in a directionopposite the one in which it ran before, ie the rotor moves clockwise.Accordingly the ratchet 20 turns the ratchet wheel 18 and thus drivesthe shaft 14 and theAworrn gear 22 and the cam wheel 23, so that the cam25 gets thrown out of engagement with the micro-switch 27 whichaccordingly closes. Now, the foot switch 40 can be released without themotor stopping, the motor getting now current through the connection 45,the micro-switch 27 and the connection 58. Now the motor cannot stopuntil the cam 25 has made one turn so that it gets once more intoengagement with the micro-switch 27. However, even this circumstance isnot sufiicient to stop the motor. The cam wheel 10 rotates also and hasgreater velocity. Most of the time the cam 24 is accordingly out ofengagement with the micro-switch 26, which is closed most of the timeand allows current to pass through the motor, through the connection 50,the contact 57, the contact 36, the connection 56 and the connection 58.For this reason the motor is kept running in a direction which causesthe rotor to move clockwise until the cam 24 of the cam wheel 10 has gotinto engagement with the micro-switch 26. Thus, it is the sharperbreaking by means of this faster moved cam which determines the point atwhich the motor 7 stops and not the less sharp breaking by means of theengagement of the cam 25 with the micro-switch 27.

As it appears from what has been said above the apparatus works in themanner intended by activating only the foot contacts 39 and 40. If, forinstance, the transmission ratio between the cam wheel 10 and the camwheel 23 is 1:10, pressing of the foot switch 39 would cause a unitvolume of the liquid contained in the container 30 to be sucked in.When, next, the foot switch 40 is pressed totally 10 unit volumes offluid consisting of 1 volume of the liquid sucked in from the container30 and 9 volumes diluting agent will be delivered to the container 30.In this manner a progressive-dilution series with the dilution ratio1:10 is attained. If a progressive dilution series in which the dilutionratio is 1:5, i.e. 2:10 is required, the foot switch 39 must be pressedtwice before the foot switch 40 and so on.

The volume of the conduit 6 depends on its length and said volume canthus always be so adjusted that it surpasses sufiiciently the number ofunit volumes which it can be required to suck into the conduit to obtaina required dilution ratio.

The magnitude of the unit volume depends on the sectional area of theconduit 6 and can be adjusted merely by replacing the conduit withanother conduit having a different cross-section but the same wallthickness,

The velocity of pumping should be so adjusted that a thorough mixing ofthe liquid to be diluted with the diluting agent is attained at thedelivery of the liquid to the container 30. A means for thus adjustingthe pumping velocity is to use a suitable velocity of the rotor shaft.-Asuitable velocity may for instance be attained by using a gear motorrunning 60 revolutions per minute, which motor may be a synchronousmotor. Hereby it is attained that the motor stops immediately onbreaking the current and that the velocity is always the same. By way ofexample of a suitable motor may be mentioned the Burgermotor designatedRSM provided with gear E.

I claim:

1. Apparatus for producing progressive-dilution series comprising avolumetric pump inserted in a conduit one end of which is connected to acontainer for diluting agent, the other end being free and adapted to belowered into basic fluid or diluted liquid, as the case may be,contained in a container for such liquid, means for activating the saidpump in one or the opposite direction and means for automaticallydiscontinuing such pumping after the transportation of a unit volumewhen the transportation takes place in direction towards the containerfor diluent, and means for automatically discontinuing transportationafter a plurality of unit volumes have been transported whentransportation takes place in the direction towards the said free end ofthe conduit.

2. Apparatus according to claim 1 in which the volume of the conduitincluding the pump space surpasses the unit volume.

3. Apparatus according to claim 1 in which the volume of the conduitincluding the pump space surpasses a plurality of unit volumes.

4. Apparatus according to claim 1, in which the pump is a flexible-hosepump the rotor of which is driven by a motor, the direction of rotationof which is shiftable.

5. Apparatus according to claim 4, in which the means for discontinuingthe pumping action consist of cams activating electric switches forbreaking the circuit of the motor, said cams being carried by cam wheelsin transmission engagement with the shaft of the motor of the flexiblehose pump, one cam wheel being carried by the rotor shaft and the otherby an auxiliary cam wheel shaft being in one-way engagement with therotor shaft and driven a plurality of times slower than the said rotorshaft.

6. A method of producing a progressive dilution series containing anumber of solutions in concentrations that are consecutive members of ageometrical progression which comprises maintaining a supply zone ofbasic solution from which a progressive dilution series is to beproduced, pumping one or more unit volumes of basic solution from saidsupply zone, simultaneously pumping in the same a corresponding amountof diluent to a reserve zone for diluent, transferring the one or morevolumes of basic solution pumped from the supply zone to a receiving 5zone for receiving diluted solution, now reverse pumping thepredetermined number of unit volumes into the receiving zone for dilutedsolution, then pumping out the two liquids in the last mentioned zone tomix them, collecting the mixed solutions, and then repeating the statedcycle of operations on further amounts of liquids.

7. The method of claim 6 in which a single pumping system is utilized inreverse pumping operation.

References Cited r FRED C. MATTERN, JR., Primary Examiner.

WILBUR J. GOODLIN, Assistant Examiner.

